Genes encoding plant adenosine 5′-phosphosulfate reductase

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a sulfate assimilation protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the sulfate assimilation protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the sulfate assimilation protein in a transformed host cell.

This application claims the benefit of U.S. Provisional Application No.60/092,833, filed Jul. 14, 1998.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingsulfate assimilation proteins in plants and seeds.

BACKGROUND OF THE INVENTION

Sulfate assimilation is the process by which environmental sulfur isfixed into organic sulfur for use in cellular metabolism. The two majorend products of this process are the essential amino acids cysteine andmethionine. These amino acids are limiting in food and feed; they cannotbe synthesized by animals and thus must be acquired from plant sources.Increasing the level of these amino acids in feed products is thus ofmajor economic value. Key to that process is increasing the level oforganic sulfur available for cysteine and methionine biosynthesis.

Multiple enzymes are involved in sulfur assimilation. These include:High affinity sulfate transporter and low affinity sulfate transporterproteins which serve to transport sulfur from the outside environmentacross the cell membrane into the cell (Smith et al. (1995) PNAS92(20):9373-9377). Once sulfur is in the cell sulfateadenylyltransferase (ATP sulfurylase) (Bolchia et al. (1999) Plant. Mol.Biol. 39(3):527-537) catalyzes the first step in assimilation,converting the inorganic sulfur into an organic form, adenosine-5′phospho-sulfate (APS). Next, several enzymes further modify organicsulfur for use in the biosynthesis of cysteine and methionine. Forexample, adenylylsulfate kinase (APS kinase), catalyzes the conversionof APS to the biosynthetic intermediate PAPS (3′-phospho-adenosine-5′phosphosulfate) (Arz et al. (1994) Biochim. Biophy. Acta1218(3):447-452). APS reductase (5′ adenylyl phosphosulphate reductase)is utilized in an alternative pathway, resulting in an inorganic butcellularly bound (bound to a carrier), form of sulfur (sulfite) (Setyaet al. (1996) PNAS 93(23):13383-13388). Sulfite reductase furtherreduces the sulfite, still attached to the carrier, to sulfide andserine O-acetyltransferase converts serine to O-acetylserine, which willserve as the backbone to which the sulfide will be transferred to fromthe carrier to form cysteine (Yonelcura-Sakakibara et al. (1998) J.Biolchem. 124(3):615-621 and Saito et al. (1995) J. Biol. Chem.270(27):16321-16326).

As described, each of these enzymes is involved in sulfate assimilationand the pathway leading to cysteine biosynthesis, which in turn servesas an organic sulfur donor for multiple other pathways in the cell,including methionine biosynthesis. Together or singly these enzymes andthe genes that encode them have utility in overcoming the sulfurlimitations known .to exist in crop plants. It may be possible tomodulate the level of sulfur containing compounds in the cell, includingthe nutritionally critical amino acids cysteine and methionine.Specifically, their overexpression using tissue specific promoters willremove the enzyme in question as a possible limiting step, thusincreasing the potential flux through the pathway to the essential aminoacids. This will allow the engineering of plant tissues with increaseslevels of these amino acids, which now often must be added a supplementsto animal feed.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding sulfate assimilation proteins. Specifically, this inventionconcerns an isolated nucleic acid fragment encoding an APS reductase andan isolated nucleic acid fragment that is substantially similar to anisolated nucleic acid fragment encoding an APS reductase. In addition,this invention relates to a nucleic acid fragment that is complementaryto the nucleic acid fragment encoding APS reductase. An additionalembodiment of the instant invention pertains to a polypeptide encodingall or a substantial portion of an APS reductase.

In another embodiment, the instant invention relates to a chimeric geneencoding an APS reductase, or to a chimeric gene that comprises anucleic acid fragment that is complementary to a nucleic acid fragmentencoding an APS reductase, operably linked to suitable regulatorysequences, wherein expression of the chimeric gene results in productionof levels of the encoded protein in a transformed host cell that isaltered (i.e., increased or decreased) from the level produced in anuntransformed host cell.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding an APSreductase, operably linked to suitable regulatory sequences. Expressionof the chimeric gene results in production of altered levels of theencoded protein in the transformed host cell. The transformed host cellcan be of eukaryotic or prokaryotic origin, and include cells derivedfrom higher plants and microorganisms. The invention also includestransformed plants that arise from transformed host cells of higherplants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of an APS reductase in a transformedhost cell comprising:

a) transforming a host cell with a chimeric gene comprising a nucleicacid fragment encoding an APS reductase; and b) growing the transformedhost cell under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of altered levels of APS reductase in the transformed hostcell.

An addition embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or a substantial portionof an amino acid sequence encoding an APS reductase.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIGS. 1A, 1B and 1C show a comparison of the amino acid sequences setforth in SEQ ID NOs:2, 4, 6, 8 and 10 and the Catharanthus roseus andArabidopsis thaliana sequences (SEQ ID NOs:11 and 12) respectively.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Sulfate Assimilation Proteins SEQ ID NO: Protein CloneDesignation (Nucleotide) (Amino Acid) APS reductase Contig composed of:1 2 chp2.pk0008.e7 p0014.ctusu54rb APS reductase ids.pk0004.f12 3 4 APSreductase se4.11g09 5 6 APS reductase sl2.pk0064.g4 7 8 APS reductasewle1.pk0005.d6 9 10

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “nucleic acid fragment” is a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. A nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize, under stringent conditions(0.1×SSC. 0.1% SDS, 65° C.), with the nucleic acid fragments disclosedherein.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASARGENE bioinformatics computing suite (DNASTAR Inc., Madison. Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, asequence of ten or more contiguous amino acids or thirty or morecontiguous nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 30 or more contiguous nucleotides maybe used in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12 or more nucleotides may be used as amplificationprimers in PCR in order to obtain a particular nucleic acid fragmentcomprising the primers. Accordingly, a “substantial portion” of anucleotide sequence comprises a nucleotide sequence that will affordspecific identification and/or isolation of a nucleic acid fragmentcomprising the sequence. The instant specification teaches amino acidand nucleotide sequences encoding polypeptides that comprise one or moreparticular plant proteins. The skilled artisan, having the benefit ofthe sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for purposes known to those skilledin this art. Accordingly, the instant invention comprises the completesequences as reported in the accompanying Sequence Listing, as well assubstantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) MolecularBiotechnology 3:225). The “3′ non-coding sequences” refer to nucleotidesequences located downstream of a coding sequence and includepolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The use of different 3′ non-coding sequences isexemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the mRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of a sulfateassimilation protein have been isolated and identified by comparison ofrandom plant cDNA sequences to public databases containing nucleotideand protein sequences using the BLAST algorithms well known to thoseskilled in the art. The nucleic acid fragments of the instant inventionmay be used to isolate cDNAs and genes encoding homologous proteins fromthe same or other plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other APS reductase enzymes, either as cDNAsor genomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, the entire sequences canbe used directly to synthesize DNA probes by methods known to theskilled artisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673; Loh et al. (1989) Science 243:217). Productsgenerated by the 3′ and 5′ RACE procedures can be combined to generatefull-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of APS reductase in those cells. This enzyme isinvolved in sulfate assimilation and the pathway leading to cysteinebiosynthesis, which in turn serves as an organic sulfur donor formultiple other pathways in the cell, including methionine biosynthesis.This enzyme and the gene(s) that encodes the protein has utility inovercoming the sulfur limitations known to exist in crop plants. It maybe possible to modulate the level of sulfur containing compounds in thecell, including the nutritionally critical amino acids cysteine andmethionine. Specifically, their overexpression using tissue specificpromoters will remove the enzyme in question as a possible limitingstep, thus increasing the potential flux through the pathway to theessential amino acids. This will allow the engineering of plant tissueswith increases levels of these amino acids, which now often must beadded a supplements to animal feed.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can thenconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:241 1-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA. Northern analysis of mRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100: 1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppresion technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded sulfate assimilation protein. An example of a vector forhigh level expression of the instant polypeptides in a bacterial host isprovided (Example 6).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181)in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4(1):37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide. Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Research5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 114(2):95-96), polymorphism of PCR-amplifiedfragments (CAPS, Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997)Nature Genetics 7:22-28) and Happy Mapping (Dear and Cook (1989) NucleicAcid Res. 17:6795-6807). For these methods, the sequence of a nucleicacid fragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402: Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149; Bensen et al. (1995) Plant Cell7:75). The latter approach may be accomplished in two ways. First, shortsegments of the instant nucleic acid fragments may be used in polymerasechain reaction protocols in conjunction with a mutation tag sequenceprimer on DNAs prepared from a population of plants in which Mutatortransposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries: Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from various corn, impatiens, soybeanand wheat tissues were prepared. The characteristics of the librariesare described below.

TABLE 2 cDNA Libraries from Corn, Impatients, Soybean and Wheat LibraryTissue Clone chp2 Corn (Zea mays L.) 11 day old leaf treated (24 hrs)with chp2.pk0008.e7 herbicides (A and B)* ids Impatiens balsaminadeveloping seed ids.pk0004.f12 p0014 Corn (Zea mays L.) leaf: plant 3 fttall, leaf 7 and leaf 8 p0014.ctusu54rb se4 Soybean (Glycine max L.)embryo, 19 days after flowering se4.11g09 sl2 Soybean (Glycine max L.)two week old developing sl2.pk0064.g4 seedlings treated with 2.5 ppmchlorimuron** wle1 Wheat (Triticum aestivum L.) leaf 7 day old etiolatedseedling wle1.pk0005.d6 *herbicide descriptions: A: Application of2-[(2,4-dihydro-2,6,9-trimethyl[1]benzothiopyrano[4,3-c]pyrazol-8-yl)carbonyl]-1,3-cyclohexanedioneS,S-dioxide; synthesis and methods of using this compound are describedin WO 97/19087, incorporated herein by reference. B: Application of2-[(2,3-dihydro-5,8-dimethylspiro[4H-1-benzothiopyran-4,2′-[1,3]dioxolan]-6-yl)carbonyl]-1,3-cyclohexanedioneS,S-dioxide; also named2-[(2,3-dihydro-5,8-dimethylspiro[4H-1-benzothiopyran-4,2′-[1,3]dioxolan]-6-yl)carbonyl]-3-hydroxy-2-cyclohexen-1-oneS,S-dioxide; synthesis and methods of using this compound are describedin WO 97/01550, incorporated herein by reference **Chlorimuron iscommercially available from Signa Chemical Co.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP* XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP* XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651). Theresulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescentsequencer.

Example 2

Identification of cDNA Clones

cDNA clones encoding sulfate assimilation proteins were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI.For convenience, the P-value (probability) of observing a match of aCDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding APS Reductase

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to APSreductase from Catharanthus roseus (NCBI Identifier No. gi 1488043) andArabidopsis thaliana (NCBI Identifier No. gi 2738756). Shown in Table 3are the BLAST results for individual ESTs (“EST”), the sequences of theentire cDNA inserts comprising the indicated cDNA clones (“FIS”), orcontigs assembled from two or more ESTs (“Contig”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toCatharanthus roseus and Arabidopsis thaliana APS Reductase Clone StatusBLAST pLog Score Contig composed of: Contig  163.00 (gi 1488043)chp2.pk0008.e7 p0014.ctusu54rb ids.pk0004.f12 FIS  139.00 (gi 1488043)se4.11g09 FIS >254.00 (gi 1488043) sl2.pk0064.g4 FIS >254.00 (gi2738756) wle1.pk0005.d6 FIS >254.00 (gi 2738756)

FIGS. 1A, 1B and 1C present an alignment of the amino acid sequences setforth in SEQ ID NOs:2, 4, 6, 8 and 10 and the Catharanthus roseus andArabidopsis thaliana sequences (SEQ ID NOs:11 and 12 respectively). Thedata in Table 4 represents a calculation of the percent identity of theamino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8 and 10 and theCatharanthus roseus and Arabidopsis thaliana sequences (SEQ ID NOs:11and 12).

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toCatharanthus roseus and Arabidopsis thaliana APS Reductase SEQ ID NO.Percent Identity to 2 79% (gi 1488043) 4 79% (gi 1488043) 6 75% (gi1488043) 8 71% (gi 2738756) 10 66% (gi 2738756)

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASARGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of an APS reductase. These sequencesrepresent the first corn, impatiens, soybean and wheat sequencesencoding APS reductase.

Example 4 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML 103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the CDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS 1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrohacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 6 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 250.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

12 1 1215 DNA Zea mays unsure (273) n = a, c, g or t 1 tgttcccggacgcgagcgag gtgcaggagc tggtgcgcac caagggcctc ttctccttct 60 acgaggacggccaccaggag tgctgccggg tgcgcaaggt gcggcccctg cgcagggcgc 120 tcaaggggcttagggcatgg atcaccggcc agaggaaaga ccagtccccc ggcaccaggg 180 ccagcatccccattgtccag gttgatcctt ccttcgaagg cctggatggc ggggccggta 240 gcttggtcaagtggaacccc gtggccaacg tcnacggcaa ggacatctgg actttcctgc 300 ggaccatggacgtacctgtc aacaccctgc atgctcaggg ctacgtgtcc atcgggtgcg 360 agccgtgcaccaggcccgtc ctgccggggc agcacgagcg tgaaggccgg tggtggtggg 420 aggacgccaaggccaaggag tgcggcctcc acaagggcaa cattgacaag gacgcccagg 480 cggcggcccccaggtccgcc aacggcaacg gctcggcggg cgccccggac atcttcgaga 540 gccccgccgtggtgtccctc acccgcaccg ggatcgagaa cctgctgcgc ctggagaacc 600 gcgccgagccgtggctcgtg gtgctgtacg cgccctggtg cccgttctgc caggccatgg 660 aggcctcctacgtggagctg gccgagaagc tggcggggtc cggggtgaag gtggccaagt 720 tccgcgcggacggcgagcag aagccgttcg cgcaggccga gctgcagctg cagagctttc 780 ccaccgtgctcctgttcccg ggccgcaccg ccaggcccat caagtacccg tcggagaaga 840 gggacgtcgactcgctcctc gccttcgtca acagcctccg gtgagagacg acctccagtg 900 agcgagaaccatcgttctct gtcagtctgt atgatcttat gttggtcttt atgagtttat 960 ctaggttcgtagagaaggga ggtggagggg actgggtttg gtagtgacac aggaaggaac 1020 gagggttcaggggggaaaaa tcaggtgtag cttttgtaac tgcaaaatga ttgcagcatg 1080 tatacctgaagtctgagctt ctgaggccct gtgcttggta gctgagggag aggttacttg 1140 tgtgtgcttatagtcagtgg cgagtgccta ctataaggtt caccggtcat ctaaagcact 1200 gttgtaaactgtatt 1215 2 293 PRT Zea mays UNSURE (91) Xaa = any amino acid 2 Phe ProAsp Ala Ser Glu Val Gln Glu Leu Val Arg Thr Lys Gly Leu 1 5 10 15 PheSer Phe Tyr Glu Asp Gly His Gln Glu Cys Cys Arg Val Arg Lys 20 25 30 ValArg Pro Leu Arg Arg Ala Leu Lys Gly Leu Arg Ala Trp Ile Thr 35 40 45 GlyGln Arg Lys Asp Gln Ser Pro Gly Thr Arg Ala Ser Ile Pro Ile 50 55 60 ValGln Val Asp Pro Ser Phe Glu Gly Leu Asp Gly Gly Ala Gly Ser 65 70 75 80Leu Val Lys Trp Asn Pro Val Ala Asn Val Xaa Gly Lys Asp Ile Trp 85 90 95Thr Phe Leu Arg Thr Met Asp Val Pro Val Asn Thr Leu His Ala Gln 100 105110 Gly Tyr Val Ser Ile Gly Cys Glu Pro Cys Thr Arg Pro Val Leu Pro 115120 125 Gly Gln His Glu Arg Glu Gly Arg Trp Trp Trp Glu Asp Ala Lys Ala130 135 140 Lys Glu Cys Gly Leu His Lys Gly Asn Ile Asp Lys Asp Ala GlnAla 145 150 155 160 Ala Ala Pro Arg Ser Ala Asn Gly Asn Gly Ser Ala GlyAla Pro Asp 165 170 175 Ile Phe Glu Ser Pro Ala Val Val Ser Leu Thr ArgThr Gly Ile Glu 180 185 190 Asn Leu Leu Arg Leu Glu Asn Arg Ala Glu ProTrp Leu Val Val Leu 195 200 205 Tyr Ala Pro Trp Cys Pro Phe Cys Gln AlaMet Glu Ala Ser Tyr Val 210 215 220 Glu Leu Ala Glu Lys Leu Ala Gly SerGly Val Lys Val Ala Lys Phe 225 230 235 240 Arg Ala Asp Gly Glu Gln LysPro Phe Ala Gln Ala Glu Leu Gln Leu 245 250 255 Gln Ser Phe Pro Thr ValLeu Leu Phe Pro Gly Arg Thr Ala Arg Pro 260 265 270 Ile Lys Tyr Pro SerGlu Lys Arg Asp Val Asp Ser Leu Leu Ala Phe 275 280 285 Val Asn Ser LeuArg 290 3 1210 DNA Impatiens balsamia 3 gcacgaggta catgttccct gatgcaattgtagtacaagg attagtaaga accaaaggac 60 tgttctcttt ctacgaagac ggacatcaagagtgctgccg cgtcagaaaa gtgaggccac 120 tgaggcgtgc tctcaagggt ctccgcgcttggatcacggg gcaaagaaaa gaccagtcgc 180 cgggaacgag atcggagatc ccagtcgtccaagtggatcc ctcttttgag ggattggttg 240 gtggagaggg tagcctggtg aagtggaatccgctggctaa tgtagatggt cgtgatgtat 300 ggaatttcct ccgagctatg aatgtgcctgttaatgcact tcatagccag ggttatgtct 360 cgattgggtg cgaaccgtgc acccgaccggtgttacctgg gcaacatgag agagaaggca 420 ggtggtggtg ggaggatgct gcggctaaggagtgtggcct acataaagga aatataaagg 480 atgccaatgg gaatggggtt gctcaagctgagggaggaga aggaactgtt acggatgctg 540 atatttttga atccaagaat gtggtgacactgagtagaag cgggattgag aatctgtcga 600 aacttcagga gaggaaagag ccatggatcgtggtcctgta tgcaccttgg tgccagttct 660 gccagggtat ggaaaaatca tacttggaattggctgaaaa gctggcggtg agcggtggtg 720 gtgtgaaggt agggaaattc cgggcagatggtgcagaaaa ggagtttgct caccaagaat 780 tgcagctggg gagctttcca acaatactcttcttccccaa acactcatct aaagccatca 840 agtacccatc tgagaaaagg gacgtggagtcattgttggc ttttgtgaac gcactcagat 900 gaatcaactg cagaaaccta gcagagctcaactggattgt tgagttcata atgctttgac 960 gaatccaata aaacacccac ccgcccctgttgtaagatgg tcagttagtc tctgttctgt 1020 tgtggcttgg tggccagagt tttggttacgtaaaaggtag ctagcaactc agaagagtcc 1080 gttttggttt cattttcttc ttcttttttgtttgattgat ttggttaaca taaaagctat 1140 cagttgttta aaccaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200 aaaaaaaaaa 1210 4 299 PRTImpatiens balsamia 4 Thr Arg Tyr Met Phe Pro Asp Ala Ile Val Val Gln GlyLeu Val Arg 1 5 10 15 Thr Lys Gly Leu Phe Ser Phe Tyr Glu Asp Gly HisGln Glu Cys Cys 20 25 30 Arg Val Arg Lys Val Arg Pro Leu Arg Arg Ala LeuLys Gly Leu Arg 35 40 45 Ala Trp Ile Thr Gly Gln Arg Lys Asp Gln Ser ProGly Thr Arg Ser 50 55 60 Glu Ile Pro Val Val Gln Val Asp Pro Ser Phe GluGly Leu Val Gly 65 70 75 80 Gly Glu Gly Ser Leu Val Lys Trp Asn Pro LeuAla Asn Val Asp Gly 85 90 95 Arg Asp Val Trp Asn Phe Leu Arg Ala Met AsnVal Pro Val Asn Ala 100 105 110 Leu His Ser Gln Gly Tyr Val Ser Ile GlyCys Glu Pro Cys Thr Arg 115 120 125 Pro Val Leu Pro Gly Gln His Glu ArgGlu Gly Arg Trp Trp Trp Glu 130 135 140 Asp Ala Ala Ala Lys Glu Cys GlyLeu His Lys Gly Asn Ile Lys Asp 145 150 155 160 Ala Asn Gly Asn Gly ValAla Gln Ala Glu Gly Gly Glu Gly Thr Val 165 170 175 Thr Asp Ala Asp IlePhe Glu Ser Lys Asn Val Val Thr Leu Ser Arg 180 185 190 Ser Gly Ile GluAsn Leu Ser Lys Leu Gln Glu Arg Lys Glu Pro Trp 195 200 205 Ile Val ValLeu Tyr Ala Pro Trp Cys Gln Phe Cys Gln Gly Met Glu 210 215 220 Lys SerTyr Leu Glu Leu Ala Glu Lys Leu Ala Val Ser Gly Gly Gly 225 230 235 240Val Lys Val Gly Lys Phe Arg Ala Asp Gly Ala Glu Lys Glu Phe Ala 245 250255 His Gln Glu Leu Gln Leu Gly Ser Phe Pro Thr Ile Leu Phe Phe Pro 260265 270 Lys His Ser Ser Lys Ala Ile Lys Tyr Pro Ser Glu Lys Arg Asp Val275 280 285 Glu Ser Leu Leu Ala Phe Val Asn Ala Leu Arg 290 295 5 1795DNA Glycine max 5 ttcggcacga gatctactct ctattttcct agcttagatt ccttctccaatggctcttgc 60 cgtttccact acttcttcct cttcagctgc agcagcagca gcagcgtcgagctctttctt 120 ctcgcgcctt ggatcttcat cggacgctaa agctccgcaa attggttcctttcggtttcc 180 ggagaggcct caagtttcgt ctggtgttgt taatttaact caaagacgctcctcggtgag 240 gccactcaat gccgaaccgc aacggaatga ttctgttgtt cctcttgcagcaactatcgt 300 tgctcctgag gttgagaagg agaaagaaga ttttgagcaa ttagcgaaagaccttgaaaa 360 ttcatctcct cttgagatta tggataaggc cctcgagaaa tttgggaacgacatcgctat 420 tgcctttagt ggtgctgaag atgttgcttt gattgagtat gcacatttgacgggtcgacc 480 ctacagagtg tttagtcttg acactgggag actgaaccca gaaacctacaaattttttga 540 cgctgttgag aagcattatg gaattcatat tgagtacatg ttccctgatgcggttgaggt 600 tcaggcatta gtaagaacta aggggctctt ctcattttac gaggatgggcatcaagagtg 660 ctgtagagta agaaaggtga ggcccttgag gagagccctt aagggtctcaaagcatggat 720 tactggacag agaaaagacc agtctcctgg tactaggtct gaaatccctattgtccaggt 780 tgatcctgtt tttgagggac tggatggtgg aattggcagc ctggtgaagtggaacccggt 840 tgcaaatgtt aatggtctag acatatggaa cttccttagg accatgaatgttcctgtaaa 900 ttcattgcat tcccaaggat atgtttcgat tggctgtgag ccatgcacaaggccggtttt 960 acccggacaa catgaaagag aaggaaggtg gtggtgggag gatgccaaagccaaggagtg 1020 tggtcttcac aaaggtaatt tgaaacagga agatgctgcc cagcttaatggaaatgggac 1080 ctcccaagga aatggctctg ccactgttgc tgacattttc atctcccagaatgtggtcag 1140 cttgagcagg tccgggattg agaatttggc aaaattagag aaccgaaaagaacactggct 1200 tgttgtgctc tatgcaccat ggtgccgctt ctgtcaggct atggaggagtcgtatgttga 1260 tctggcagag aagttagcaa ggtcaggagt gaaggttgca aaattcagagccgatggaga 1320 gcagaaggaa tatgcaaaga gtgaactgca gttgggaagc ttccccacaatacttctctt 1380 ccccaagcac tcttctcaac caattaagta cccttcagaa aagagagatgttgattcatt 1440 gacggcattc gtgaatgcct tacggtgatg gtcaattgag tatcttgctcaatgttccgt 1500 cgtaccatac cggcaataaa tttcttcaca gatttgggca attcactgaaaatgggaatg 1560 gcagtttttg atagcaaaac gaagattctc agctagcagt atccctgtatagatttagat 1620 aaccttcctc acaaatataa ttgtagtagt catgaggagg atgtgatttcctgttttgtt 1680 agatgagtag agttatggtt gtattatgtt gtttcttcac tatcataatctactttttta 1740 gattttgcca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaa 1795 6 472 PRT Glycine max 6 Met Ala Leu Ala Val Ser Thr Thr SerSer Ser Ser Ala Ala Ala Ala 1 5 10 15 Ala Ala Ala Ser Ser Ser Phe PheSer Arg Leu Gly Ser Ser Ser Asp 20 25 30 Ala Lys Ala Pro Gln Ile Gly SerPhe Arg Phe Pro Glu Arg Pro Gln 35 40 45 Val Ser Ser Gly Val Val Asn LeuThr Gln Arg Arg Ser Ser Val Arg 50 55 60 Pro Leu Asn Ala Glu Pro Gln ArgAsn Asp Ser Val Val Pro Leu Ala 65 70 75 80 Ala Thr Ile Val Ala Pro GluVal Glu Lys Glu Lys Glu Asp Phe Glu 85 90 95 Gln Leu Ala Lys Asp Leu GluAsn Ser Ser Pro Leu Glu Ile Met Asp 100 105 110 Lys Ala Leu Glu Lys PheGly Asn Asp Ile Ala Ile Ala Phe Ser Gly 115 120 125 Ala Glu Asp Val AlaLeu Ile Glu Tyr Ala His Leu Thr Gly Arg Pro 130 135 140 Tyr Arg Val PheSer Leu Asp Thr Gly Arg Leu Asn Pro Glu Thr Tyr 145 150 155 160 Lys PhePhe Asp Ala Val Glu Lys His Tyr Gly Ile His Ile Glu Tyr 165 170 175 MetPhe Pro Asp Ala Val Glu Val Gln Ala Leu Val Arg Thr Lys Gly 180 185 190Leu Phe Ser Phe Tyr Glu Asp Gly His Gln Glu Cys Cys Arg Val Arg 195 200205 Lys Val Arg Pro Leu Arg Arg Ala Leu Lys Gly Leu Lys Ala Trp Ile 210215 220 Thr Gly Gln Arg Lys Asp Gln Ser Pro Gly Thr Arg Ser Glu Ile Pro225 230 235 240 Ile Val Gln Val Asp Pro Val Phe Glu Gly Leu Asp Gly GlyIle Gly 245 250 255 Ser Leu Val Lys Trp Asn Pro Val Ala Asn Val Asn GlyLeu Asp Ile 260 265 270 Trp Asn Phe Leu Arg Thr Met Asn Val Pro Val AsnSer Leu His Ser 275 280 285 Gln Gly Tyr Val Ser Ile Gly Cys Glu Pro CysThr Arg Pro Val Leu 290 295 300 Pro Gly Gln His Glu Arg Glu Gly Arg TrpTrp Trp Glu Asp Ala Lys 305 310 315 320 Ala Lys Glu Cys Gly Leu His LysGly Asn Leu Lys Gln Glu Asp Ala 325 330 335 Ala Gln Leu Asn Gly Asn GlyThr Ser Gln Gly Asn Gly Ser Ala Thr 340 345 350 Val Ala Asp Ile Phe IleSer Gln Asn Val Val Ser Leu Ser Arg Ser 355 360 365 Gly Ile Glu Asn LeuAla Lys Leu Glu Asn Arg Lys Glu His Trp Leu 370 375 380 Val Val Leu TyrAla Pro Trp Cys Arg Phe Cys Gln Ala Met Glu Glu 385 390 395 400 Ser TyrVal Asp Leu Ala Glu Lys Leu Ala Arg Ser Gly Val Lys Val 405 410 415 AlaLys Phe Arg Ala Asp Gly Glu Gln Lys Glu Tyr Ala Lys Ser Glu 420 425 430Leu Gln Leu Gly Ser Phe Pro Thr Ile Leu Leu Phe Pro Lys His Ser 435 440445 Ser Gln Pro Ile Lys Tyr Pro Ser Glu Lys Arg Asp Val Asp Ser Leu 450455 460 Thr Ala Phe Val Asn Ala Leu Arg 465 470 7 1629 DNA Glycine max 7gcacgaggag agaacccata acagctagtt aatggccctc gctttcactt cttcaatttc 60cgcaccaact tccaccttcc catcatcgga acccaaactt ccgcaaattg ggtcaattag 120gatttcggag aggcccattg gaggcgccgt taatttcaat ttatctcaaa gacggagctt 180ggtaaagccc gttaacgccg aacctccacg caaggattcc attgttcctc tcgcagcaac 240aaccatcgtt gcttctgctt ctgagacgaa agaggaagat tttgaacaga tagccagtga 300tctcgacaat gcttcacctc ttgaaatcat ggatagagcc ctcgacaaat tcggcaacga 360catagctatt gccttcagtg gtgctgaaga tgttgctttg attgagtatg cgaaattgac 420gggtcgaccc tttagggttt tcagtttgga cactgggaga ctgaacccag aaacttatca 480actttttgat gcggttgaga agcattatgg aattcgcatt gagtacatgt tccctgatgc 540tgttgaggtt caggcattgg tgaggagtaa ggggttattc tctttctacg aggatgggca 600ccaagagtgt tgcagggtga gaaaggtgag gcctttaagg agggccctta agggtctcag 660agcatggata actggtcaga ggaaagacca gtcacctggt actaggtctg aaataccggt 720tgttcaggtt gatccggctt ttgagggaat ggatggtgga attggaagct tggtgaagtg 780gaaccctgtt gcaaatgtga agggccatga catatggaac ttccttagga ccatgaatgt 840gcctgtgaat tccttgcatg caaaaggata tgtttccatt gggtgtgagc cctgcactag 900gcctgtttta cctgggcaac atgaaaggga agggaggtgg tggtgggagg atgccaaagc 960taaggaatgt ggtcttcaca aaggaaatgt aaagcagcag aaagaggagg atgttaatgg 1020aaatgggcta tcccaatccc atgcaaatgg tgatgctacc actgtgcctg acattttcaa 1080cagcccgaat gtagttaact tgagcaggac tggaattgag aatttggcaa aattggagga 1140ccgaaaggaa ccatggcttg ttgtgcttta tgcaccatgg tgcccctact gccaggctat 1200ggaggaatct tatgttgact tagcagacaa gttagcaggg tcaacaggga tgaaggttgg 1260aaaatttaga gcagatggag aacagaaaga atttgcaaag agtgaactgc aattgggaag 1320cttccctacg atattatttt tcccaaagca ttcgtctcgg ccaacaataa agtatccctc 1380agaaaagaga gatgttgatt ccttgatggc atttgtaaat gccttaagat gaggatatca 1440ggaaattttc ttcgtttttg ggttgcaatt ccactttgac tatacgtaca gcgggttcct 1500tctttatgct attacgtgta tataccattc gtttacagat tcttctgtga actcgttgga 1560agtgggaatg gaggtttata caaataagat actcagtttt gaatggtttt aaaaaaaaaa 1620aaaaaaaaa 1629 8 466 PRT Glycine max 8 Met Ala Leu Ala Phe Thr Ser SerIle Ser Ala Pro Thr Ser Thr Phe 1 5 10 15 Pro Ser Ser Glu Pro Lys LeuPro Gln Ile Gly Ser Ile Arg Ile Ser 20 25 30 Glu Arg Pro Ile Gly Gly AlaVal Asn Phe Asn Leu Ser Gln Arg Arg 35 40 45 Ser Leu Val Lys Pro Val AsnAla Glu Pro Pro Arg Lys Asp Ser Ile 50 55 60 Val Pro Leu Ala Ala Thr ThrIle Val Ala Ser Ala Ser Glu Thr Lys 65 70 75 80 Glu Glu Asp Phe Glu GlnIle Ala Ser Asp Leu Asp Asn Ala Ser Pro 85 90 95 Leu Glu Ile Met Asp ArgAla Leu Asp Lys Phe Gly Asn Asp Ile Ala 100 105 110 Ile Ala Phe Ser GlyAla Glu Asp Val Ala Leu Ile Glu Tyr Ala Lys 115 120 125 Leu Thr Gly ArgPro Phe Arg Val Phe Ser Leu Asp Thr Gly Arg Leu 130 135 140 Asn Pro GluThr Tyr Gln Leu Phe Asp Ala Val Glu Lys His Tyr Gly 145 150 155 160 IleArg Ile Glu Tyr Met Phe Pro Asp Ala Val Glu Val Gln Ala Leu 165 170 175Val Arg Ser Lys Gly Leu Phe Ser Phe Tyr Glu Asp Gly His Gln Glu 180 185190 Cys Cys Arg Val Arg Lys Val Arg Pro Leu Arg Arg Ala Leu Lys Gly 195200 205 Leu Arg Ala Trp Ile Thr Gly Gln Arg Lys Asp Gln Ser Pro Gly Thr210 215 220 Arg Ser Glu Ile Pro Val Val Gln Val Asp Pro Ala Phe Glu GlyMet 225 230 235 240 Asp Gly Gly Ile Gly Ser Leu Val Lys Trp Asn Pro ValAla Asn Val 245 250 255 Lys Gly His Asp Ile Trp Asn Phe Leu Arg Thr MetAsn Val Pro Val 260 265 270 Asn Ser Leu His Ala Lys Gly Tyr Val Ser IleGly Cys Glu Pro Cys 275 280 285 Thr Arg Pro Val Leu Pro Gly Gln His GluArg Glu Gly Arg Trp Trp 290 295 300 Trp Glu Asp Ala Lys Ala Lys Glu CysGly Leu His Lys Gly Asn Val 305 310 315 320 Lys Gln Gln Lys Glu Glu AspVal Asn Gly Asn Gly Leu Ser Gln Ser 325 330 335 His Ala Asn Gly Asp AlaThr Thr Val Pro Asp Ile Phe Asn Ser Pro 340 345 350 Asn Val Val Asn LeuSer Arg Thr Gly Ile Glu Asn Leu Ala Lys Leu 355 360 365 Glu Asp Arg LysGlu Pro Trp Leu Val Val Leu Tyr Ala Pro Trp Cys 370 375 380 Pro Tyr CysGln Ala Met Glu Glu Ser Tyr Val Asp Leu Ala Asp Lys 385 390 395 400 LeuAla Gly Ser Thr Gly Met Lys Val Gly Lys Phe Arg Ala Asp Gly 405 410 415Glu Gln Lys Glu Phe Ala Lys Ser Glu Leu Gln Leu Gly Ser Phe Pro 420 425430 Thr Ile Leu Phe Phe Pro Lys His Ser Ser Arg Pro Thr Ile Lys Tyr 435440 445 Pro Ser Glu Lys Arg Asp Val Asp Ser Leu Met Ala Phe Val Asn Ala450 455 460 Leu Arg 465 9 1827 DNA Triticum aestivum 9 gcacgaggttaaaacacatt tgccagctcc gacaaacatc cctgcgaatt tgagagggag 60 gaagggttcattcagcggcc ggtaatcaat ggcttccgct actgcttcca tctcgtcgca 120 ctccatcgccctgcgcgatc tcaaagccgc gaggattgga gccgtgaggc agcaggtggc 180 cgtggttcctgcgggcctgc cggcaacggc gcccaagggc cagcgcgcga gggcggtgcg 240 cccgctgtgcgcggcggagc cagcgaggaa gccagtgtcg gcctccgcgg cctcgtcgcc 300 ggtggcgccggtggaggagg aggcatctgc cgtggcggcc gtggactacg aggccctggc 360 gcaggagctggtgggcgcgt cgccgctgga gatcatggat cgtgcgctcg acatgttcgg 420 ctccgaaatcgccatcgcct tcagtggtgc cgaggacgtg gccctcatcg aatacgcgaa 480 actgactggacgccccttca gggtgttcag ccttgacact gggcgactga acccagagac 540 atacgaactcttcgacaagg tggagaagca ctatggtatc cacatcgagt acatgttccc 600 tgaggccagcgaggtgcaag accttgtgag gagcaagggc ctcttctctt tctacgagga 660 cggacaccaggagtgctgca gggtgaggaa ggttcggccc ttgaggaggg ccctcaaggg 720 cctcaaggcctggatcaccg ggcagaggaa ggatcagtcc cctggcacca gagccagcat 780 ccctgttgttcaagttgatc cgtcttttga agggctggat ggtggagccg gtagcttgat 840 caagtggaaccctgtggcta atgtggatgg caaggatatc tggaccttcc tcaggaccat 900 ggatgtccctgtgaacaccc tgcatgctca aggctacgtc tccattgggt gcgagccgtg 960 caccaggcccgtgttgccgg ggcagcacga gagggaaggg aggtggtggt gggaggacgc 1020 cacggccaaggagtgcggcc tgcacaacgg taacatcgac aaggaaggtc aggcacccaa 1080 ggtcggcgtcaacggcaacg gctcggccga ggccagcgcc ccagacatct tccagagcca 1140 ggccatcgtcaacctcaccc gtcccgggat cgagaacctc ctgcggctcg agaaccgcgc 1200 cgagccgtggctcaccgtcc tctacgctcc ctggtgccca tactgccagg caatggaggc 1260 gtcctacgttgagctggccg agaagctgag cggctcaggc atcaaggtgg ccaagttccg 1320 cgcggacggcgagcagaagc cattcgcgca ggcggagctg caactacaga gcttcccgac 1380 gatcctcctgttccccggcc gcaccgtgaa gcccatcaag tacccgtccg agaagaggga 1440 cgtccagtccctcctcgcct tcgtgaacag cctcagatga gtggtcagag aaccggagaa 1500 ccatcgttctctgcattggt accggcggtg tctaggcatt attatgtagt ggtagcgaga 1560 gaggatggatcaacggaaat gttggagaca gaggagtgtg gggacgcagg gacagcggct 1620 caaagcccctccattataag ggggtgggga tttgtgtgta gttgtagcta gatgtttgta 1680 aggaagttcaaataagagta ctagttttga aattttgatc caaggcttca tcgagagttt 1740 ggacaatatactcgtggttc actcggtcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800 aaaaaaaaaaaaaaaaaaaa aaaaaaa 1827 10 463 PRT Triticum aestivum 10 Met Ala Ser AlaThr Ala Ser Ile Ser Ser His Ser Ile Ala Leu Arg 1 5 10 15 Asp Leu LysAla Ala Arg Ile Gly Ala Val Arg Gln Gln Val Ala Val 20 25 30 Val Pro AlaGly Leu Pro Ala Thr Ala Pro Lys Gly Gln Arg Ala Arg 35 40 45 Ala Val ArgPro Leu Cys Ala Ala Glu Pro Ala Arg Lys Pro Val Ser 50 55 60 Ala Ser AlaAla Ser Ser Pro Val Ala Pro Val Glu Glu Glu Ala Ser 65 70 75 80 Ala ValAla Ala Val Asp Tyr Glu Ala Leu Ala Gln Glu Leu Val Gly 85 90 95 Ala SerPro Leu Glu Ile Met Asp Arg Ala Leu Asp Met Phe Gly Ser 100 105 110 GluIle Ala Ile Ala Phe Ser Gly Ala Glu Asp Val Ala Leu Ile Glu 115 120 125Tyr Ala Lys Leu Thr Gly Arg Pro Phe Arg Val Phe Ser Leu Asp Thr 130 135140 Gly Arg Leu Asn Pro Glu Thr Tyr Glu Leu Phe Asp Lys Val Glu Lys 145150 155 160 His Tyr Gly Ile His Ile Glu Tyr Met Phe Pro Glu Ala Ser GluVal 165 170 175 Gln Asp Leu Val Arg Ser Lys Gly Leu Phe Ser Phe Tyr GluAsp Gly 180 185 190 His Gln Glu Cys Cys Arg Val Arg Lys Val Arg Pro LeuArg Arg Ala 195 200 205 Leu Lys Gly Leu Lys Ala Trp Ile Thr Gly Gln ArgLys Asp Gln Ser 210 215 220 Pro Gly Thr Arg Ala Ser Ile Pro Val Val GlnVal Asp Pro Ser Phe 225 230 235 240 Glu Gly Leu Asp Gly Gly Ala Gly SerLeu Ile Lys Trp Asn Pro Val 245 250 255 Ala Asn Val Asp Gly Lys Asp IleTrp Thr Phe Leu Arg Thr Met Asp 260 265 270 Val Pro Val Asn Thr Leu HisAla Gln Gly Tyr Val Ser Ile Gly Cys 275 280 285 Glu Pro Cys Thr Arg ProVal Leu Pro Gly Gln His Glu Arg Glu Gly 290 295 300 Arg Trp Trp Trp GluAsp Ala Thr Ala Lys Glu Cys Gly Leu His Asn 305 310 315 320 Gly Asn IleAsp Lys Glu Gly Gln Ala Pro Lys Val Gly Val Asn Gly 325 330 335 Asn GlySer Ala Glu Ala Ser Ala Pro Asp Ile Phe Gln Ser Gln Ala 340 345 350 IleVal Asn Leu Thr Arg Pro Gly Ile Glu Asn Leu Leu Arg Leu Glu 355 360 365Asn Arg Ala Glu Pro Trp Leu Thr Val Leu Tyr Ala Pro Trp Cys Pro 370 375380 Tyr Cys Gln Ala Met Glu Ala Ser Tyr Val Glu Leu Ala Glu Lys Leu 385390 395 400 Ser Gly Ser Gly Ile Lys Val Ala Lys Phe Arg Ala Asp Gly GluGln 405 410 415 Lys Pro Phe Ala Gln Ala Glu Leu Gln Leu Gln Ser Phe ProThr Ile 420 425 430 Leu Leu Phe Pro Gly Arg Thr Val Lys Pro Ile Lys TyrPro Ser Glu 435 440 445 Lys Arg Asp Val Gln Ser Leu Leu Ala Phe Val AsnSer Leu Arg 450 455 460 11 463 PRT Catharanthus roseus 11 Met Ala LeuAla Phe Thr Ser Ser Thr Ala Ile His Gly Ser Leu Ser 1 5 10 15 Ser SerPhe Glu Gln Thr Lys Ala Ala Ala Ala Gln Phe Gly Ser Phe 20 25 30 Gln ProLeu Asp Arg Pro His Thr Ile Ser Pro Ser Val Asn Val Ser 35 40 45 Arg ArgArg Leu Ala Val Lys Pro Ile Asn Ala Glu Pro Lys Arg Asn 50 55 60 Glu SerIle Val Pro Ser Ala Ala Thr Thr Val Ala Pro Glu Val Glu 65 70 75 80 GluLys Val Asp Val Glu Asp Tyr Glu Lys Leu Ala Asp Glu Leu Gln 85 90 95 AsnAla Ser Pro Leu Glu Ile Met Asp Lys Ser Leu Ala Lys Phe Gly 100 105 110Asn Asp Ile Ala Ile Ala Phe Ser Gly Ala Glu Asp Val Ala Leu Ile 115 120125 Glu Tyr Ala His Leu Thr Gly Arg Pro Phe Arg Val Phe Ser Leu Asp 130135 140 Thr Gly Arg Leu Asn Pro Glu Thr Tyr Lys Phe Phe Asp Thr Val Glu145 150 155 160 Lys Gln Tyr Gly Ile His Ile Glu Tyr Met Phe Pro Asp AlaVal Glu 165 170 175 Val Gln Ala Leu Val Arg Ser Lys Gly Leu Phe Ser PheTyr Glu Asp 180 185 190 Gly His Gln Glu Cys Cys Arg Val Arg Lys Val ArgPro Leu Arg Arg 195 200 205 Ala Leu Lys Gly Leu Arg Ala Trp Ile Thr GlyGln Arg Lys Asp Gln 210 215 220 Ser Pro Gly Thr Arg Ser Glu Ile Pro ValVal Gln Val Asp Pro Val 225 230 235 240 Phe Glu Gly Met Asp Gly Gly ValGly Ser Leu Val Lys Trp Asn Pro 245 250 255 Val Ala Asn Val Glu Gly LysAsp Ile Trp Asn Phe Leu Arg Ala Met 260 265 270 Asp Val Pro Val Asn ThrLeu His Ser Gln Gly Tyr Val Ser Ile Gly 275 280 285 Cys Glu Pro Cys ThrArg Pro Val Leu Pro Gly Gln His Glu Arg Glu 290 295 300 Gly Arg Trp CysTrp Glu Asp Ala Lys Ala Lys Glu Cys Gly Leu His 305 310 315 320 Lys GlyAsp Ile Lys Glu Gly Thr Leu Ile Ile Trp Asp Gly Ala Val 325 330 335 AsnGly Asn Gly Ser Asp Thr Ile Ala Asp Ile Phe Asp Thr Asn Asn 340 345 350Val Thr Ser Leu Ser Arg Pro Gly Ile Glu Asn Leu Leu Lys Leu Glu 355 360365 Glu Arg Arg Glu Ala Trp Leu Val Val Leu Tyr Ala Pro Trp Cys Arg 370375 380 Phe Cys Gln Ala Met Glu Gly Ser Tyr Leu Glu Leu Ala Glu Lys Leu385 390 395 400 Ala Gly Ser Gly Val Lys Val Gly Lys Phe Lys Ala Asp GlyAsp Gln 405 410 415 Lys Ala Phe Ala Gln Gln Glu Leu Gln Leu Asn Ser SerPro Thr Ile 420 425 430 Leu Phe Phe Pro Lys His Ser Ser Lys Pro Ile LysTyr Pro Ser Glu 435 440 445 Lys Arg Asp Val Asp Ser Leu Met Ala Phe ValAsn Ala Leu Arg 450 455 460 12 465 PRT Arabidopsis thaliana 12 Met AlaMet Ser Val Asn Val Ser Ser Ser Ser Ser Ser Gly Ile Ile 1 5 10 15 AsnSer Arg Phe Gly Val Ser Leu Glu Pro Lys Val Ser Gln Ile Gly 20 25 30 SerLeu Arg Leu Leu Asp Arg Val His Val Ala Pro Val Ser Leu Asn 35 40 45 LeuSer Gly Lys Arg Ser Ser Ser Val Lys Pro Leu Asn Ala Glu Pro 50 55 60 LysThr Lys Asp Ser Met Ile Pro Leu Ala Ala Thr Met Val Ala Glu 65 70 75 80Ile Ala Glu Glu Val Glu Val Val Glu Ile Glu Asp Phe Glu Glu Leu 85 90 95Ala Lys Lys Leu Glu Asn Ala Ser Pro Leu Glu Ile Met Asp Lys Ala 100 105110 Leu Glu Lys Tyr Gly Asn Asp Ile Ala Ile Ala Phe Ser Gly Ala Glu 115120 125 Asp Val Ala Leu Ile Glu Tyr Ala His Leu Thr Gly Arg Pro Phe Arg130 135 140 Val Phe Ser Leu Asp Thr Gly Arg Leu Asn Pro Glu Thr Tyr ArgPhe 145 150 155 160 Phe Asp Ala Val Glu Lys His Tyr Gly Ile Arg Ile GluTyr Met Phe 165 170 175 Pro Asp Ser Val Glu Val Gln Gly Leu Val Arg SerLys Gly Leu Phe 180 185 190 Ser Phe Tyr Glu Asp Gly His Gln Glu Cys CysArg Val Arg Lys Val 195 200 205 Arg Pro Leu Arg Arg Ala Leu Lys Gly LeuLys Ala Trp Ile Thr Gly 210 215 220 Gln Arg Lys Asp Gln Ser Pro Gly ThrArg Ser Glu Ile Pro Val Val 225 230 235 240 Gln Val Asp Pro Val Phe GluGly Leu Asp Gly Gly Val Gly Ser Leu 245 250 255 Val Lys Trp Asn Pro ValAla Asn Val Glu Gly Asn Asp Val Trp Asn 260 265 270 Phe Leu Arg Thr MetAsp Val Pro Val Asn Thr Leu His Ala Ala Gly 275 280 285 Tyr Ile Ser IleGly Cys Glu Pro Cys Thr Lys Ala Val Leu Pro Gly 290 295 300 Gln His GluArg Glu Gly Arg Trp Trp Trp Glu Asp Ala Lys Ala Lys 305 310 315 320 GluCys Gly Leu His Lys Gly Asn Val Lys Glu Asn Ser Asp Asp Ala 325 330 335Lys Val Asn Gly Glu Ser Lys Ser Ala Val Ala Asp Ile Phe Lys Ser 340 345350 Glu Asn Leu Val Thr Leu Ser Arg Gln Gly Ile Glu Asn Leu Met Lys 355360 365 Leu Glu Asn Arg Lys Glu Pro Trp Ile Val Val Leu Tyr Ala Pro Trp370 375 380 Cys Pro Phe Cys Gln Ala Met Glu Ala Ser Tyr Asp Glu Leu AlaAsp 385 390 395 400 Lys Leu Ala Gly Ser Gly Ile Lys Val Ala Lys Phe ArgAla Asp Gly 405 410 415 Asp Gln Lys Glu Phe Ala Lys Gln Glu Leu Gln LeuGly Ser Phe Pro 420 425 430 Thr Ile Leu Val Phe Pro Lys Asn Ser Ser ArgPro Ile Lys Tyr Pro 435 440 445 Ser Glu Lys Arg Asp Val Glu Ser Leu ThrSer Phe Leu Asn Leu Val 450 455 460 Arg 465

What is claimed is:
 1. An isolated polynucleotide comprising: (a) anucleotide sequence encoding a polypeptide having adenosine5′-phosphosulfate reductase activity, wherein the polypeptide has anamino acid sequence of at least 95% sequence identity, based on theClustal V method of alignment, when compared to SEQ ID NO:6, or (b) acomplement of the nucleotide sequence of (a), wherein the complement andthe nucleotide sequence consist of the same number of nucleotides andare 100% complementary.
 2. The polynucleotide of claim 1, wherein theamino acid sequence of the polypeptide comprises SEQ ID NO:6.
 3. Thepolynucleotide of claim 1 wherein the nucleotide sequence comprises SEQID NO:5.
 4. A vector comprising the polynucleotide of claim
 1. 5. Arecombinant DNA construct comprising the polynucleotide of claim 1operably linked to at least one regulatory sequence.
 6. A method fortransforming a cell, comprising introducing into a cell the recombinantDNA construct of claim
 5. 7. A cell comprising the recombinant DNAconstruct of claim
 5. 8. A method for producing a transgenic plantcomprising transforming a plant cell with the recombinant DNA constructof claim 5 regenerating a plant from the transformed plant cell.
 9. Aplant comprising the recombinant DNA construct of claim
 5. 10. A seedcomprising the recombinant DNA construct of claim
 5. 11. A method forthe production of a polypeptide having adenosine 5′-phosphosulfatereductase activity comprising the steps of cultivating the cell of claim7 under conditions that allow for the synthesis of the polypeptide andisolating the polypeptide from the cultivated cells, from culturemedium, or from both the cultivated cells and the culture medium.